Institute for Maternal and Child Health-IRCCS "Burlo Garofolo", Trieste, Italy.
Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.
PLoS One. 2021 Mar 5;16(3):e0247603. doi: 10.1371/journal.pone.0247603. eCollection 2021.
The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.
CRISPR/Cas9 细菌系统已被证明是几种生物遗传操作的强大工具，但同源直接修复（HDR）的序列替换效率远低于随机插入/缺失（indel）的产生。许多研究集中于使用双 sgRNA、细胞同步周期和具有合理设计的单链寡核苷酸（ssODN）来提高 HDR 效率。在这项研究中，我们评估了这三种方法的协同作用以提高 HDR 效率。对于我们的测试，我们选择 TNFα 基因（NM_000594），因为它在各种生物学过程和疾病中具有关键作用。我们的结果首次表明，使用具有非对称供体设计的两个 sgRNA 和三次转染事件，如何将 HDR 效率从无法检测到的 HDR 事件显著提高到 39%的 HDR 效率，并为促进 CRISPR/Cas9 介导的人类基因组编辑提供了一种新策略。此外，我们证明了 TNFα 基因座可以用 CRISPR/Cas9 方法进行编辑，这为将来安全地纠正每个患者的特定突变提供了机会。
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